Insect Pathology

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Dedication

We dedicate this second edition of Insect Pathology to Dr. Lawrence (Lerry) A. Lacey in recognition of his outstanding contributions to insect pathology and microbial control. Lerry received his PhD from the University of California in Riverside in 1978, and has been at the forefront of fundamental and applied research with various entomopathogens, including baculoviruses, bacteria, fungi, and nematodes to control key insect pests of agricultural, medical, and veterinary importance. He has distinguished himself nationally and internationally with his numerous scientific achievements and has mentored a number of students and international scientists. Internationally, his accomplishments include (1) the use of Bacillus thuringiensis israelensis for the control of the black fly, Simulium damnosum, the principal vector of onchocerciasis in West Africa, (2) basic and applied research to select the most appropriate microbial control agents for the suppression of the Japanese beetle, Popillia japonica, in the Azores, Portugal, and (3) foreign exploration and research on fungal entomopathogens of the whitefly, Bemisia tabaci type B. Nationally, his comprehensive studies on the granulovirus of the codling moth have highlighted the utility of this pathogen in organic and conventional apple and pear orchards and revealed various factors that influence its efficacy. His research has also shown that nematodes can be used effectively to control the overwintering stage of the codling moth in fruit bins. In addition to more than 140 peer-reviewed publications, he has edited Manual of Techniques in Insect Pathology and co-edited Safety of Microbial Insecticides and Field Manual of Techniques in Invertebrate Pathology, which are widely used throughout the world. Lerry was designated a fellow of the Entomological Society of America in 2008 and has served in many different capacities for the Society for Invertebrate Pathology and the Entomological Society of America. He is the quintessential insect pathologist and microbial control researcher. We honor Lerry for his many noteworthy scientific accomplishments, but beyond these achievements, he has contributed in many other ways to insect pathology and microbial control. As a leader and spokesperson, he has brought integrity and wisdom to insect pathology and microbial control and has helped growers to implement the use of microbial control agents for controlling insect pests.

Preface

Insect pathology is an essential component of entomology and provides a non-chemical alternative for insect pest management. There are several groups of organisms that can infect and kill insects, including viruses, fungi, microsporidia, bacteria, protists, and nematodes. The dilemma in insect pathology has been that even though there has been a steady increase in our knowledge of insect pathogens, their share of the market for insect control remains minuscule. Why is this? Is it perhaps due to a lack of understanding on specific aspects of the pathogens, e.g., their ecology in the field? Or is the current state of our knowledge – even after more than 100 years of research – a limiting factor in making these microbes effective, economically acceptable, and widely accepted?

The twentieth and early twenty-first centuries have brought incredible advances to the field of insect pathology. From the landmark books by Edward Steinhaus (Chapters 1 and 2) and his founding in 1959 of a scientific journal fully devoted to insect and invertebrate pathology, which to date has published over 5000 peer-reviewed articles, we have seen the development of molecular biology concepts and tools as the most revolutionary scientific breakthrough of our era. These advances have led to the sequencing of hundreds of genomes, including those of various viruses, bacteria, and fungi of importance to insect pathology (Chapter 2). Molecular biology has also led to the elucidation of phylogenetic relatedness that has clearly challenged many of our basic assumptions, such as in the placement of microsporidia in the kingdom Fungi.

Our discipline has moved very rapidly since the publication of Insect Diseases, edited by G. E. Cantwell (1974), Insect Pathology by Y. Tanada and H. K. Kaya (1993), Principles of Insect Pathology by D. G. Boucias and J. C. Pendland (1998), and the second edition of Controle Microbiano de Insetos, edited by Sérgio Batista Alves (1998). A number of other edited books in insect and invertebrate pathology have also been published with a focus on more specific areas, such as those edited or co-edited by L. A. Lacey, to whom this second edition is dedicated. The vast amount of new concepts and technologies, as well as the enormous complexity of insect pathogens, make it nearly impossible for any specific scientist to master the field and write a book covering the topic. The large database has led to this edited second edition of Insect Pathology, which consists of 13 chapters. The book should satisfy the needs of insect pathologists, entomologists, mycologists, nematologists, protistologists, ecologists, biological control researchers, and students interested in all aspects of insect pathology in a single up-to-date information source. It should also serve as a resource for others in any of the biological sciences who are interested in insect pathology

We thank Y. Tanada for allowing the use of the title of the book as a second edition of Insect Pathology and all the authors for their tireless efforts in contributing to the book. All royalties from this book will be donated to the Chris J. Lomer Memorial Fund, which supports scientists from developing countries to attend the Society for Invertebrate Pathology annual meetings. We express our deepest appreciation to the following chapter reviewers: T. G. Andreadis, J. S. Cory, D. Cox-Foster, M. S. Goettel, G. Hoch, M. S. Hunter, A. Koppenhöfer, J. C. Lord, and R.  D. Possee. Special thanks to Pat Gonzalez and Kristi Gomez at Elsevier for their help and support throughout this project. Thanks also to Ann Simpkins for meticulously cross-checking references in each chapter without losing her good sense of humor. Finally, F. E. Vega and H. K. Kaya are most grateful to Wendy S. Higgins, Ian G. Vega, and Joanne Kaya for their loving support.

Fernando E. Vega

Harry K. Kaya

References

1. Biblioteca de Ciências Agrárias Luiz de Queiroz Vol. 4. Alves SB, ed. Controle Microbiano de Insetos. 1998; Piracicaba.

2. Boucias DG, Pendland JC. Principles of Insect Pathology. Boston: Kluwer Academic; 1998.

3. Insect Diseases. Vols. 1 and 2. Cantwell GE, ed. New York: Marcel Dekker; 1974.

4. Tanada Y, Kaya HK. Insect Pathology. San Diego: Academic Press; 1993.

Foreword

Insect pathology is a biological discipline that is unique in many ways. Pathology, the study of diseases and disorders, is an ancient pursuit at least with respect to human body, dating back almost 25 centuries to Hippocrates, father of Western medicine. Discovering what has gone awry in a body has been key to figuring out ways of fixing the problems in a wide range of organisms. Thus, there are veterinary pathologists and plant pathologists dedicated to identifying causes of diseases with the ultimate goal of opening a path to restoring their non-human animal and photosynthesizing organisms of interest, respectively, to good health. But insect pathologists are, for want of a better phrase, uniquely bipolar with respect to their subjects; whereas many share the goal with other kinds of pathologists of finding ways to right whatever has gone wrong, the vast majority of insect pathologists are dedicated to making things go wrong, notably by identifying disease-causing agents that will dispatch their subjects quickly, efficiently, and specifically.

Underlying the bipolar nature of the discipline is the tremendous diversity of the taxon comprising the targets of study. With over 900,000 species described to date, it is not at all surprising that insects interact with humans in more different ways than perhaps any other group of organisms. Some insects, such as the domesticated silkworm or western honey bee, provide goods and services upon which entire economies depend; others, however, ravage crops, destroy buildings, spread deadly pathogens, and destroy nations. The tremendous diversity of ways in which humans interact with insects is another dimension of insect pathology that distinguishes it from other biological disciplines: its almost unimaginable complexity.

But the complexity of the human–insect interaction masks even greater complexity that must be addressed by insect pathologists. Each of those 900,000-plus species hosts its own unique microflora and fauna that can influence its health and welfare, so the insect pathologist literally has millions of combinations and permutations of organisms. A diverse range of invertebrate taxa, including many insect species, is infected with obligate rickettsial endosymbionts in the genus Wolbachia; entomopathogenic nematodes depend on their symbiotic bacteria to infect and kill their hosts. Thus, insect pathologists not only have to become expert entomologists, they also have to become microbiologists to differentiate between pathogens that destroy their hosts and symbionts that keep them alive. And traditional microbiological training will not do: the various and sundry pathogens that cause disease in insects, including viruses, fungi, bacteria, protists, and nematodes, and virtually every other kind of disease-causing organism, for the most part are specific to insects and have no effect on organisms other than insects. This arrangement works well for designing pest controls with minimal impacts on non-target species but it also requires insect pathologists to develop their own body of knowledge and their own methods and techniques, rather than relying on comparative pathology for enlightenment.

Because an important component of insect pathology, particularly in the context of biological control of insect pests, is the ability to predict outbreaks and epidemics, insect pathologists also to some extent have to become ecologists. Understanding the forces influencing insect population fluctuations – density, weather, other environmental factors – provides valuable insights into disease dynamics and helps to inform forecasting and modeling. Knowledge about insect physiology and toxicology helps the pathologist to understand the origins and progression of diseases, and a basic knowledge of evolution is essential for identifying mechanisms underlying differential resistance and susceptibility. An insect pathologist, then, is a little bit of just about every kind of life scientist.

Despite its status as a specialized field (that requires broad training), insect pathology has contributed to improving the quality of human life in ways that are disproportionate to the number of its practitioners throughout history. It is clear that people benefit when agricultural pests can be stopped by disease, but the benefits of insect pathology to agriculture have been magnified with molecular methods – today, for example, 63% of American corn and 73% of American cotton is genetically engineered to produce an insecticidal toxin from an insect-specific bacterium described by insect pathologists. And the impact of insect pathology extends well beyond the farm. In some respects, insect pathology was a necessary component for refining the germ theory of disease: the legendary French microbiologist Louis Pasteur, charged with stopping pébrine, a mysterious epidemic that was destroying France's silkworms, provided the first practical demonstration that killing germs (a microsporidian parasite, in the case of pébrine) could cure disease. Insect pathologists, in the course of studying viruses that kill insects, gained insights that led them to devise remarkably efficient way of genetically altering these so-called baculoviruses and co-opt them for manufacturing all kinds of recombinant proteins, today used to manufacture a dizzying diversity of human therapeutics, including, most recently, vaccines to protect people against bird flu.

It would seem, given the wide-ranging scope of the field and seemingly limitless applications of the knowledge it generates, that becoming an insect pathologist could be insuperably challenging. Fortunately, the editors of this volume are not easily daunted; every page helps to untangle the complexity and to illuminate the amazing interactions among insects, microbes, and humans. Fernando E. Vega and Harry K. Kaya are to be commended for putting together a book that is not only enormously useful to those specifically involved in the field but also incredibly fascinating even to those with no stake in either lengthening the life expectancy of useful insects or shortening the life expectancy of pestiferous ones.

May R. Berenbaum

Contributors

Numbers in parentheses indicate the pages on which the authors' contributions begin.

James J. Becnel, (133, 221), Center for Medical, Agricultural & Veterinary Entomology, United States Department of Agriculture, Agricultural Research Service, Gainesville, Florida 32608, USA

May R. Berenbaum, (xv), Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

Meredith Blackwell, (171), Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803, USA

Denny J. Bruck, (29), Horticulture Crops Research Laboratory, United States Department of Agriculture, Agricultural Research Service, Corvallis, OR 97330, USA

Yan Ping (Judy) Chen, (133), Bee Research Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA

David J. Clarke, (395), Department of Microbiology and Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland

Elizabeth W. Davidson, (13), Arizona State University, School of Life Sciences, Tempe, Arizona 85287, USA

Robert Harrison, (73), Invasive Insect Biocontrol and Behavior Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA

Kelli Hoover, (73), Department of Entomology, Pennsylvania State University, University Park, Pennsylvania 16802, USA

Grant L. Hughes, (351), College of Agricultural Sciences, Department of Entomology, Pennsylvania State University, University Park, Pennsylvania 16802, USA

Trevor A. Jackson, (265), AgResearch, Biocontrol and Biosecurity, Lincoln Research Centre, Canterbury, New Zealand

Rosalind R. James, (425), Pollinating Insects – Biology, Management and Systematics Research, United States Department of Agriculture, Agricultural Research Service, Logan, Utah 84322, USA

Juan Luis Jurat-Fuentes, (265, 461), Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, Tennessee 37996, USA

Harry K. Kaya, (1), Department of Nematology, University of California, Davis, California 95616, USA

Lawrence A. Lacey, (29), IP Consulting International, Yakima, Washington 98908, USA

Carlos E. Lange, (367), Comisión de Investigaciones Científicas (CIC) de la provincia de Buenos Aires CCT La Plata, CEPAVE-CONICET–UNLP, La Plata 1900, Argentina

Edwin E. Lewis, (395), Department of Entomology and Department of Nematology, University of California, Davis, California 95616, USA

Zengzhi Li, (425), Department of Forestry, Anhui Agricultural University, Hefei, Anjui 230036, PR China

Jeffrey C. Lord, (367), Stored Product Insect Research Unit, Grain Marketing & Production Research Center, United States Department of Agriculture, Agricultural Research Service, Manhattan, Kansas 66502, USA

Janet Jennifer Luangsa-ard, (171), Phylogenetics Laboratory, BIOTEC, Thailand Science Park, Khlong Luang, Pathum Thani 12120, Thailand

Jonathan G. Lundgren, (461), North Central Agricultural Research Laboratory, United States Department of Agriculture, Agricultural Research Service, Brookings, South Dakota 57006, USA

Nicolai V. Meyling, (171), Department of Ecology, University of Copenhagen, Frederiksberg C, DK-1871, Denmark

David H. Oi, (221), Center for Medical, Agricultural & Veterinary Entomology, United States Department of Agriculture, Agricultural Research Service, Gainesville, Florida 32608, USA

Jason L. Rasgon, (351), College of Agricultural Sciences, Department of Entomology, Pennsylvania State University, University Park, Pennsylvania 16802, USA

David I. Shapiro-Ilan, (29), United States Department of Agriculture, Agricultural Research Service, Byron, Georgia 31008, USA

Leellen F. Solter, (221), Illinois Natural History Survey, Prairie Research Institute, University of Illinois, Champaign, Illinois 61801, USA

Steven M. Valles, (133), Center for Medical, Agricultural & Veterinary Entomology, United States Department of Agriculture, Agricultural Research Service, Gainesville, Florida 32608, USA

Fernando E. Vega, (1, 171), Sustainable Perennial Crops Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland 20705, USA

Table of Contents

Cover Image

Title

Copyright

Dedication

Preface

Foreword

Contributors

Chapter 1. Scope and Basic Principles of Insect Pathology

1.1 Introduction

1.2 Categories of Disease

1.3 Basic Principles in Insect Pathology

REFERENCES

Chapter 2. History of Insect Pathology

2.1 Introduction

2.2 Early History of the Discovery of Insect Pathogens

2.3 Early Discoveries of Other Pathogens

2.4 Development of Insect Pathogens for Biological Control

2.5 Events that Brought Scientists Together and Advanced the Field

2.6 Unexpected Products of Research on Insect Pathogens

2.7 Conclusions

REFERENCES

Chapter 3. Principles of Epizootiology and Microbial Control

3.1 Introduction

3.2 Epizootiology: Basic Principles

3.3 Microbial Control (Applied Epizootiology)

3.4 Future Research Directions

REFERENCES

Chapter 4. Baculoviruses and Other Occluded Insect Viruses

4.1 Introduction

4.2 Classification and Phylogeny

4.3 Baculovirus Infection, Replication, Pathology, and Transmission

4.4 Use of Baculoviruses for Insect Pest Control

4.5 Future Research Directions

Acknowledgments

REFERENCES

Chapter 5. RNA Viruses Infecting Pest Insects

5.1 Introduction

5.2 Classification, Phylogeny, Structure, and Genome Organization

5.3 Infection, Replication, Pathology, Transmission, and Host Range

5.4 Future Research Directions

REFERENCES

Chapter 6. Fungal Entomopathogens

6.1 Introduction

6.2 Classification and Phylogeny

6.3 Biology

6.4 Ecology

6.5 Use of Fungal Entomopathogens as Biological Control Agents

6.6 Future Research Directions

Acknowledgments

REFERENCES

Chapter 7. Microsporidian Entomopathogens

7.1 Introduction

7.2 Classification and Phylogeny

7.3 Life History

7.4 Biological Control Programs: Case Histories

7.5 Future Research Directions

REFERENCES

Chapter 8. Bacterial Entomopathogens

8.1 Introduction

8.2 Classification and Phylogeny

8.3 Infection, Replication, Pathology, and Transmission

8.4 Gram-Positive Entomopathogens: Phylum Firmicutes, Class Bacilli, Order Bacillales

8.5 Gram-Negative Bacteria

8.6 Other Potentially Pathogenic Bacteria: Infection by Mollicutes

8.7 Field use: Examples, Success, and Constraints

8.8 Future Research Directions

REFERENCES

Chapter 9. Infections in Arthropod Hosts

9.1 Introduction

9.2 Classification and Phylogeny

9.3 Pathogen Versus Mutualist

9.4 Historical Overview of Wolbachia and Vector Control

9.5 Reproductive Manipulations

9.6 Pathogen Interference and Pathogen Protection

9.7 Applied Use of Wolbachia

9.8 Future Research Directions

REFERENCES

Chapter 10. Protistan Entomopathogens

10.1 Introduction

10.2 Classification and Phylogeny

10.3 Associations, Signs, and Symptoms

10.4 Amoebozoa

10.5 Apicomplexa

10.6 Ciliophora

10.7 Euglenozoa

10.8 Helicosporidia

10.9 Future Research Directions

REFERENCES

Chapter 11. Nematode Parasites and Entomopathogens

11.1 Introduction

11.2 Classification and Phylogeny

11.3 Insect-Parasitic Nematodes

11.4 Entomopathogenic Nematodes: Monoxenous, Lethal Parasites with Symbionts

11.5 Biodiversity

11.6 Infective Stage Juvenile and Bacterial Storage

11.7 Infection

11.8 Nutrition Within the Insect

11.9 Natural Populations and Host Associations

11.10 Aging and Lifespan

11.11 Survival Mechanisms

11.12 Future Research Directions

REFERENCES

Chapter 12. From Silkworms to Bees

12.1 Introduction

12.2 Diseases of Silkworms

12.3 Diseases of Bees

12.4 Future Research Directions

REFERENCES

Chapter 13. Physiology and Ecology of Host Defense Against Microbial Invaders

13.1 Introduction

13.2 Behavioral and Physical Barriers to Infection

13.3 Physiological Response to Infections

13.4 Managing Resistance to Entomopathogens

13.5 Future Research Directions

REFERENCES

Index

Chapter 1

Scope and Basic Principles of Insect Pathology

Harry K. Kaya∗ and Fernando E. Vega†

∗University of California, Davis, California, USA

†United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, USA

Chapter Outline

1.1 Introduction

1.2 Categories of Disease

1.3 Basic Principles in Insect Pathology

1.3.1 Entomopathogens

1.3.2 Some Major Classification and Taxonomic Changes

1.3.3 Portal of Entry

1.3.4 Microbial Toxins

1.3.5 Infectivity

1.3.6 Pathogenicity and Virulence

1.3.7 Dosage

1.3.8 Signs, Symptoms, and Syndromes

1.3.9 Course of Infection

1.3.10 Acute, Chronic, and Latent Infections

1.3.11 Koch's Postulates

1.3.12 Diagnosis

References

Summary

Insects are the dominant animals in the world, with more than one million described species. The vast majority of insects are innocuous or beneficial to humans, but a small percentage are pests that require a significant amount of our time, effort, and funds to reduce their negative effects on food production and our health and welfare. One environmentally acceptable method to control these insect pests is to use pathogens. The study of pathogens infecting insects is referred to as insect pathology. Insect pathology is the study of anything that goes wrong with an insect and, therefore, includes non-pathogenic and pathogenic causes. The present focus is on pathogens that can be used as microbial control agents of insects. Here, the basic principles in insect pathology including the microorganisms that cause diseases, their classification and phylogeny, portal of entry, infectivity, pathogenicity and virulence, course of disease, Koch’s postulates, and diagnosis are covered.

1.1 Introduction

Insects represent three-quarters of all animal species in the world, with the vast majority of them being terrestrial and/or occurring in freshwater systems (Daly et al., 1998). More than 99% of the approximately one million described insect species (Grimaldi and Engel, 2005) are either innocuous or beneficial to humans, such as the silkworm (Bombyx mori), cochineal scale (Dactylopius coccus), pollinators, and parasitoids and predators (Gullan and Cranston, 2005; Pedigo and Rice, 2009). To place this in another context, only 1% of the known insect species are our competitors that vie for our crops and stored products, damage our belongings, serve as vectors for plant pathogens, or are of medical or veterinarian importance by feeding on us or our livestock and pets and, in some cases, serving as vectors of disease agents to humans and other vertebrates (Pedigo and Rice, 2009). Even though the number of insect pest species is small compared to the number of described insect species, they require a significant amount of our time, effort, and funds to reduce their negative effects on food production and our health and welfare.

One of the main tactics to control insect pests is the use of chemical pesticides. Unfortunately, the application of chemical pesticides can (1) have a negative effect on human health and the environment; (2) result in resistance of the pest species to pesticides; and (3) kill or negatively affect non-target organisms. An alternative to chemical control is biological control (or biocontrol), which is the study and use of living organisms for the suppression of population densities of pest insects (Eilenberg et al., 2001). The living organisms are predators, parasitoids and entomopathogens (meaning microorganisms capable of causing diseases in insects; from the Greek entoma = insect, pathos = suffering, gennaein = to produce, synonymous with insect pathogens).

The use of microorganisms for biological control is commonly referred to as microbial control, an approach that includes four strategies: classical, inoculation, inundation, and conservation (Eilenberg et al., 2001). Classical biological control involves the intentional importation, release and establishment of entomopathogens into a new environment. Inoculation biological control deals with the release of an entomopathogen with the expectation that it will multiply and will provide temporary control. This approach is sometimes referred to as augmentation biological control. Inundative biological control is dependent upon the release of significant amounts of inoculum of an entomopathogen to provide immediate control of the pest; control ensues from the release inoculum and not from its progeny. Conservation biological control involves the modification of the environment to protect and enhance an established entomopathogen. An entomopathogen can also be judiciously used with chemical pesticides in an integrated pest management program (Tanada and Kaya, 1993) or combined with a chemical substance(s) that enhances its effectiveness as a microbial control agent (Koppenhöfer et al., 2002; Koppenhöfer and Fuzy, 2008).

Before the development of microbial control agents as management tools for insect pests, maladies had been recorded from beneficial insects for a long time. Thus, the discipline of insect pathology dates back to over 2000 years ago when the Chinese recorded diseases in the silkworm and the Greeks noted diseases in the honey bee, Apis mellifera (see Chapter 2). Through the ages, many diseases have been discovered and described from both beneficial and pestiferous insects. This early development of the discipline can be attributed to (1) the curiosity of scientists in describing and ascertaining the cause of pathological conditions in insects; (2) the need to find cures for diseases that afflicted beneficial insects; and (3) the potential use of pathogens to control insect pests.

What is insect pathology? Broadly defined, insect pathology is the study of anything that goes wrong [i.e., disease (lack of ease)] with an insect. Disease is a process that represents the response of the insect’s body to insult or injury (Steinhaus, 1949, 1963c). Often, it is not easy to separate a healthy insect from one that is diseased owing to the absence of symptoms. Steinhaus (1963c) differentiates a healthy insect from a diseased one as follows: A healthy insect is one so well adjusted in its internal environment and to its external environment that is capable of carrying on the functions necessary for its maintenance, growth, and multiplication with the least expenditure of energy. A diseased insect is simply one that is not healthy; it is an insect that can no longer tolerate an injury or hardship without having an abnormal strain placed upon it.

The scope of insect pathology encompasses many subdisciplines in entomology (Fig. 1.1). In ecology, for example, epizootics of viral, bacterial, fungal, microsporidian, and nematode diseases can cause significant mortality to devastate insect populations. In physiology, biochemistry, and toxicology, diseased insects will affect the results of the experiments with differences in enzyme, lipid, or protein profiles compared to healthy insects, and diseased insects will be more susceptible than healthy insects to pesticides. Moreover, our knowledge of insect pathology has made significant contributions in (1) the control and eradication of diseases in laboratory insect colonies reared for research, sterile insect technique programs, pet food sales, educational purposes, exhibits (e.g., insect zoos), and for the sale of beneficial insects such as the silkworm and honey bee (see Chapter 12) and parasitoids and predators (Inglis and Sikorowski, 2009a, b); (2) the investigation of the intracellular (Chapter 9) and extracellular symbiosis including the disruption of mutualistic relationships between insects and microbes (Douglas, 2007, 2010); (3) the development of Bacillus thuringiensis (Bt) and other bacteria for microbial control (Chapter 8); and (4) the diagnosis or identification of etiological agents that cause insect diseases in the laboratory and field (Hukuhara, 1987; Inglis and Sikorowski, 2009b). In human medicine, baculoviruses in tissue culture systems have been used for the production of papillomavirus, influenza vaccines (Safdar and Cox, 2007; Einstein et al., 2009) and research for malaria vaccines (Blagborough et al., 2010). Steinhaus (1963c) indicated that the basic elements of insect pathology embrace etiology, pathogenesis, symptomatology, morphopathology, physiopathology, and epizootiology. Thus, insect pathology contributes to many other disciplines beyond entomology such as microbiology, veterinary and human medicine, agriculture, and basic biology.

FIGURE 1.1 Relationship between insect pathology and other subdisciplines in entomology.

1.2 Categories of Disease

Insects are exposed to a wide array of non-living (abiotic) or living (biotic) factors (i.e., causal agents) that can result in disease. The factors leading to a disease state can be referred to as non-infectious or infectious (Steinhaus, 1963c), making it clear that both types of maladies are an integral part of insect pathology. Furthermore, Steinhaus (1963c) separated the non-infectious diseases using the following categories: (1) mechanical injuries; (2) injuries caused by physical agents; (3) injuries caused by poisons or chemical agents; (4) diseases caused by nutritional disturbances or deficiencies of proper nutriments; (5) diseases caused by deranged physiology and metabolism; (6) genetic diseases or inherited abnormal conditions; (7) congenital anomalies and malformations, non-generic teratologies; (8) certain tumors and neoplasm (i.e., those not associated with microbes); (9) disturbances in development and in regenerative capacity of tissues; and (10) injuries caused by parasitization or infestation by other insects or arachnids or by predation. For more detailed information on diseases caused by non-infectious agents, the reader is referred to Steinhaus (1963a, b), Cantwell (1974), and Tanada and Kaya (1993).

Tanada and Kaya (1993) summarized the categories of insect diseases following the classification scheme used by Steinhaus (1949) that included both non-infectious and infectious diseases. These were: (1) the presence or absence of an infectious microorganism (i.e., diseases caused by infectious and non-infectious agents); (2) the extent of the disease (i.e., local, focal, or systemic disease); (3) the location or site of the disease (i.e., midgut, fat body, nerve, hemocyte, hypodermis, etc.); (4) the course of the disease (i.e., chronic, subacute, acute); (5) the source of the infectious agent (i.e., exogenous, endogenous, or idiopathic); (6) the etiological or causal agent (virus, bacterium, fungus, protist, or nematode); (7) the distribution or prevalence of the disease in an insect population (i.e., sporadic, enzootic, or epizootic); (8) the method of transmission (i.e., direct contact, vector, per os, transovum, or transovarial); and (9) the basis of sequence (i.e., primary, secondary, attenuated, progressive, mixed, or multiple). Although these categories are useful, Tanada and Kaya (1993) used the broader categories of diseases as those caused by amicrobial (non-infectious) agents and those caused by microbial (infectious) agents.

The focus of this book is on microbes that cause diseases in insects, with emphasis on their use as microbial control agents. In line with this focus, the book includes the historical development of insect pathology and microbial control (see Chapter 2), the principles of microbial control and epizootiology (Chapter 3), the various pathogen groups infecting insects (Chapters 4–8, 10, and 11), and resistance to entomopathogens (Chapter 13). It also covers Wolbachia, a genus of obligate bacteria, to control arthropods using transinfection into novel hosts (Chapter 9), and pathogens of beneficial insects, especially of silkworms and bees (Chapter 12).

1.3 Basic Principles in Insect Pathology

Basic principles in insect pathology include: (1) the microorganisms that cause diseases (entomopathogens); (2) understanding the classification and phylogeny of entomopathogens; (3) how the microorganisms invade an insect host (portal of entry); (4) whether toxins are involved in the disease process (microbial toxins); (5) infectivity of the microorganisms (infectivity); (6) the disease-producing power of the microorganisms (pathogenicity and virulence); (7) the number of microorganisms needed to cause an infection (dosage); (8) the manifestation of disease (signs, symptoms, and syndromes); (9) the progress of the infection (course of disease); (10) types of infection (acute, chronic, and latent); (11) the proof that a given microorganism is the cause of the disease (Koch’s postulates); and (12) how to determine and/or identify the causal agent (diagnosis).

The definition of a pathogen as used in this book is A microorganism capable of producing disease under normal conditions of host resistance and rarely living in close association with the host without producing disease (Steinhaus and Martignoni, 1970; also see Martignoni et al., 1984 and Onstad et al., 2006). Even though the term parasite is often used interchangeably or synonymously with pathogen, the term parasite is used in this book as defined by Onstad et al. (2006) as an organism that lives at its host’s expense, obtaining nutriment from the living substance of the latter, depriving it of useful substance, or exerting other harmful influence upon the host. The term parasitic is functionally distinct from pathogenic in that the parasite usually does not cause the mortality of the host, whereas the pathogen is routinely lethal to the host (Steinhaus and Martignoni, 1970; Onstad et al., 2006). Thus, there is a distinction between parasites and pathogens. The term entomopathogen is used in the context as defined earlier as a microorganism capable of producing a disease in insects, and will be used interchangeably with insect pathogen and pathogen. Therefore, entomopathogenic bacteria would refer to bacteria that produce diseases in their insect hosts and have the capability of being lethal to them. Yet, in the case of many entomopathogenic protists, they will infect their insect hosts but are not lethal to them. Thus, use of the term entomopathogenic can be paradoxical, and the reader should be aware of this situation. Furthermore, for nematodes (see Chapter 11), some (i.e., mermithids) are referred to as parasites as the second stage juveniles enter a host and no reproduction takes place, whereas others (i.e., steinernematids and heterorhabditids) are referred to as entomopathogens because they kill their host and reproduce (see Section 1.3.1).

1.3.1 Entomopathogens

The infectious agents, entomopathogens, are microorganisms that invade and reproduce in an insect and spread to infect other insects. These entomopathogens include non-cellular agents (viruses), prokaryotes (bacteria), eukaryotes (fungi and protists), and multicellular animals (nematodes). In the latter group, nematodes differ from the other entomopathogens by having digestive, reproductive, nervous, and excretory systems, and characteristics of parasitoids and predators. However, they have no functional response and often produce pathologies similar to other entomopathogens, and many, especially steinernematid and heterorhabditid nematodes, can invade and reproduce in an insect, and can spread to infect other insects (Kaya and Gaugler, 1993; Grewal et al., 2005).

Not all microorganisms cause infection even after they enter the insect’s hemocoel. The lack of an infection may be due to the resistant characteristics of the host or to the inability of the microbe to survive and reproduce in the host. Many entomopathogens show a high degree of specificity and will infect only one or several insect species, whereas others are generalists and infect a number of insect species in different orders and may infect species in different phyla. These infectious microorganisms can be separated into four broad categories of opportunistic, potential, facultative, and obligate pathogens, keeping in mind that they do not infect all insects. The following definitions are from Onstad et al. (2006):

• Opportunistic pathogen: A microorganism which does not ordinarily cause disease but which, under certain conditions (e.g., impaired host immunity), becomes pathogenic (e.g., Aspergillus flavus).

• Potential pathogen: 1) A microorganism that has no method of invading or infecting a host but can multiply and cause disease if it gains entrance, for example, though a wound; potential pathogens generally grow readily in culture and do not cause specific diseases in specific hosts. 2) a secondary invader (e.g., Serratia marcescens).

• Facultative pathogen: "A pathogen that can infect and multiply in host animals but is also capable of multiplying in the environment; facultative pathogens generally are readily cultured in vitro" (e.g., Bacillus thuringiensis, Beauveria bassiana).

• Obligate pathogen: "A pathogen that can multiply in nature only within the bodies of specific hosts in which it causes specific diseases. Obligate pathogens usually have a narrow host range and can be cultured in vitro only with difficulty, if at all; therefore, some mechanism must exist for their transmission from one host generation to another" (e.g., Paenibacillus popilliae, microsporidia, baculoviruses).

1.3.2 Some Major Classification and Taxonomic Changes

Major changes in the classification of some entomopathogens have occurred since the publication of the first edition of Insect Pathology by Tanada and Kaya (1993). Some of these changes include the exclusion of Oomycota from the Kingdom Fungi (see Chapter 6), the Microsporidia were in the Phylum Protozoa but are now in the Kingdom Fungi (Chapter 7), and the Protozoa were in the Kingdom Animalia and have been reclassified to the Kingdom Protista (Chapter 10). Several major reclassifications have also occurred at various levels within most of the pathogen groups (see Classification and Phylogeny in each of the pathogen chapters).

At the generic and species level, recent taxonomic revisions within various entomopathogen groups can confuse the reader not familiar with the older literature. With viruses, for example, the International Committee for the Taxonomy of Viruses (ICTV) (http://www.ictvonline.org/index.asp?bhcp=1) has adopted the following guidelines for taxonomic nomenclature of viruses. Italicization occurs in formal taxonomic usage, when the writer is explicitly referring to a taxon (e.g., family Baculoviridae or Reoviridae and genus Alphabaculovirus or Cypovirus), but no italicization is used when the viruses are referred in the vernacular (e.g., baculovirus infection, alphabaculoviruses, or cypoviruses) (see Chapters 4 and 5). A name referring to any virus species (not just the type species of a genus or family) is fully italicized. For example, references to the species Autographa californica multiple nuclepolyhedrovirus are printed in italics. In some cases, the type species uses a common or descriptive name such as Yellow fever virus (family: Flaviviridae; genus: Flavivirus) or Deformed wing virus (family: Iflaviridae; genus: Iflavirus), which is in italics (Chapters 5 and 12).

Examples from fungi include Paecilomyces fumosoroseus and P. farinosus, which have been reclassified as Isaria fumosorosea and I. farinosa, respectively; and Verticillium lecanii was reclassified into several species in the new genus Lecanicillium, which includes L. lecanii (see Chapter 6). In addition, Metarhizium (Bischoff et al., 2009) and Beauveria (Rehner et al., 2011) have undergone major changes based on phylogenetic analyses. An example of a generic reclassification within the microsporidia is the new genus Endoreticulatus replacing Pleistophora. Although Nosema is still a valid genus, a well-known species, N. locustae, used as a microbial control agent of locusts, is now Paranosema locustae.

With bacteria, some Bacillus species that are obligate pathogens of insects have been reclassified into the genus Paenibacillus. In the older literature, Bacillus popilliae and B. larvae, pathogens of scarab larvae and honey bee larvae, respectively, are now called Paenibacillus popilliae (see Chapter 8) and P. larvae (Chapter 12). In another example, the genus of the facultative mosquito pathogen, Bacillus sphaericus, has been reclassified to the genus Lysinibacillus. In this case, the older name has been retained in Chapter 8 because it is still commonly used even after the taxonomic change.

1.3.3 Portal of Entry

The portals of entry are the sites through which an entomopathogen invades or gains entry into an insect host (Fig. 1.2). The most likely portals of entry into the insect host are through the mouth (per os) or integument. The entomopathogens, especially nematodes, may also invade through the anus (per anal) or spiracles. Other routes of invasion for pathogens include through wounds or injuries to the integument, congenital passage within the ova (transovarial transmission) or on the ova (transovum transmission), and the contaminated ovipositor of parasitoids.

FIGURE 1.2 The primary portal of entry for most entomopathogens (viruses, bacteria, protists, microsporidia, and some nematodes) is primarily through the mouth ( per os ), whereas for fungi and some nematodes, the primary portal of entry is through the cuticle. The entomopathogens that enter through the mouth can infect the midgut cells and/or penetrate into the hemocoel to cause a systemic infection. Similarly, the entomopathogens that penetrate through the integument may initiate the infection on the cuticle (fungi) with subsequent penetration into the hemocoel or penetrate directly into the hemocoel (some protists and nematodes). In some cases, a few fungal species may enter a host through the mouth or nematodes may enter the anus or spiracles (not shown in figure). Once the entomopathogens reproduce in the first host, the infective propagules are released or leave from a dead or living host and can initiate a new infection in another susceptible host.

(Modified after Tanada and Kaya, 1993.)

The usual portal of entry for most entomopathogens (viruses, bacteria, protists, microsporidia, some fungi, and some nematodes), especially for insects with chewing, chewing/lapping, and sponging mouthparts, is per os, or for most entomopathogenic fungi and many nematodes, it is through the integument. For per os entry, once the entomopathogen gets into the gut, it may reproduce in the digestive lumen (e.g., bacteria) or it may penetrate through the peritrophic membrane, infect the midgut cells (e.g., viruses, bacteria, microsporidia, some fungi, and protists) and/or invade directly into the hemocoel (e.g., nematodes). Those entomopathogens that invade into the midgut cells and then into the hemocoel can infect and reproduce in specific tissues (e.g., some granuloviruses only infect the fat body), reproduce in the hemolymph (e.g., Paenibacillus popilliae), or cause a systemic infection of many different tissues (e.g., nucleopolyhedroviruses, many microsporidia, and protists). For entry through the integument, fungi use enzymes, specialized structures, and pressure to penetrate through the cuticle into the hemocoel (see Chapter 6), and nematodes use a stylet or tooth to penetrate directly into the hemocoel (Chapter 11).

Once the entomopathogen becomes established and reproduces in the insect, it usually produces a resistant stage that can survive in the environment for varying periods. The resistant stages include occlusion bodies for baculoviruses, cypoviruses, and entomopoxviruses, spores for bacteria and protistans, environmental spores for microsporidia, resting spores or sclerotia for fungi, and infective juveniles for steinernematid and heterorhabditid nematodes. These resistant stages are usually the infective stages that will infect a new host. Some non-resistant stages such as non-occluded virions, vegetative bacterial rods and fungal conidia will infect insects but do not survive long in nature. In other cases, the entomopathogen may survive in an alternate host, require an alternate host for successful completion of its life cycle, or remain in its usual host through the adult stage when it is transmitted to the next generation. Thus, a pathogen may have one or more mechanisms of survival. When a susceptible host is encountered and the pathogen is in its infectious state, it can initiate a new infection in a healthy host (Fig. 1.2).

1.3.4 Microbial Toxins

In some cases, the entomopathogen (e.g., some bacteria) does not need to infect cells or invade into the hemocoel to cause disease. It is confined to the digestive tract, produces dysentery, and causes the insect to shrink in size (brachyosis), resulting in death (Bucher, 1961; Jackson et al., 2001). In such cases, the insects appear to be affected by toxins that are produced by the bacterium. Hence, a disease can be brought about in a susceptible insect host by the pathogen through the actions of chemical or toxic substances.

Two types of toxins, catabolic and anabolic, can be produced by entomopathogens (Tanada and Kaya, 1993). Catabolic toxins result from decomposition brought about by the activity of the pathogen, whereas anabolic toxins are substances synthesized by the pathogen. The breakdown of proteins, carbohydrates, and lipids by the pathogen may produce toxic alcohols, acids, mercaptans, alkaloids, etc. In the case of anabolic toxins, the substances synthesized by the pathogen can be classified as exotoxins and endotoxins. Exotoxins are excreted or passed out of the cells of the pathogen during reproduction and have been isolated from entomopathogens, especially bacteria and fungi. Endotoxins produced by the pathogen are not excreted but confined within the cell. These endotoxins are liberated when the pathogen forms a resistant stage, dies, or degenerates. The best known endotoxin in insect pathology is produced by Bt (see Chapter 8). In this case, as the bacterium initiates the sporulation process, excess proteins are produced, resulting in the formation of a proteinaceous crystal (δ-endotoxin) within the sporangial wall adjacent to the spore. The sporangial wall is easily ruptured, releasing the δ-endotoxin and spore into the environment. The δ-endotoxin is a protoxin, and when it is placed in a high pH solution as occurs in the insect gut, the toxic component becomes activated and adversely affects the midgut cells. Thus, intoxication is brought about by the activity of the pathogen in the form of toxins.

Not all toxins produced by microorganisms are in the realm of insect pathology. For example, at least two bacteria (actinomycetes), Streptomyces avermitilis and Saccharopolyspora spinosa, produce exotoxins that have been developed into the insecticides, avermectins and spinosad, respectively. In contrast to Bt, these actinomycetes are not covered in this book because the toxins alone are used with no living pathogenic microorganisms involved. However, Bt spore or vegetative rod does not need to be present to cause the disease because the δ-endotoxin alone can kill a susceptible insect host. Although this statement appears contradictory with what was stated above for the actinomycetes, the application of Bt is with the δ-endotoxin and the spore. Transgenic plants containing the Bt δ-endotoxin, on the other hand, are not considered to be part of insect pathology per se, but are aspects of host plant resistance. Yet, with the frequent application of Bt as an organism in agricultural systems and the planting of transgenic plants with the Bt toxin genes in major agricultural crops (e.g., corn and cotton), the resistance to Bt can and has occurred even before transgenic crops were available. Accordingly, insect resistance to pathogens including managing resistance in Bt transgenic crops is covered in Chapter 13.

1.3.5 Infectivity

Infectivity is the ability of a microorganism to enter the body of a susceptible insect and produce an infection. However, many insect species harbor microorganisms that are beneficial rather than harmful. These mutualistic microorganisms infect and reproduce in the insects but are contained within specific cells (mycetocytes or bacteriocytes) or tissues (mycetomes or bacteriome) and are beneficial by producing some useful product for their insect hosts. The relationship is mutualistic because the microorganisms also benefit by obtaining nutrients and protection from their insect hosts. Thus, an infection may result in a non-diseased condition. The focus here is on the diseased condition, but the reader should be aware that many microorganisms are mutualistically associated with insects (Bourtzis and Miller, 2003, 2006, 2009). This book does include Wolbachia (Chapter 9) because they have the potential to be manipulated and used as control agents of some insect pest species.

When infection results in disease, it generally causes detectable pathological effects such as injuries or dysfunctions (i.e., impairments in function, especially of a bodily system or organ). There are two main factors associated with a disease: invasiveness and pathologies resulting in abnormalities or dysfunctions. In some cases where toxins are involved, invasiveness of the pathogen into cells and tissues or into the hemocoel need not occur.

When an infectious agent is transmitted naturally by direct contact to an insect, the resultant disease is called contagious or communicable. Contagious diseases are common in insects and occur with all major pathogen groups (i.e., viruses, bacteria, microsporidia, fungi, protists, and nematodes). Infection and contamination are not the same, as a susceptible insect may be contaminated or be harboring a pathogen without being infected. A non-susceptible insect, other organisms or objects may also be contaminated with a pathogen. In both situations, the pathogen is a potential source to infect a susceptible host.

1.3.6 Pathogenicity and Virulence

Pathogenicity and virulence are two terms used regularly in insect pathology. These two terms have spurred some debate among scientists (see Thomas and Elkinton, 2004; Shapiro-Ilan et al., 2005), but the definitions by Onstad et al. (2006) and Tanada and Kaya (1993) and recommended by Shapiro-Ilan et al. (2005) are used in this book. Pathogenicity is defined as the quality or state or being pathogenic, the potential or ability to produce disease, whereas virulence is defined as the disease producing power of an organism, the degree of pathogenicity within a group or species (Shapiro-Ilan et al., 2005). Thus, pathogenicity is a qualitative term and for a given host and pathogen, it is absolute, whereas virulence quantifies pathogenicity and is variable owing to the strain of the pathogen or to environmental effects. Furthermore, pathogenicity can be considered as an all-or-none response; that is, the microorganism is either pathogenic to a host or not and is applied to groups or species (Shapiro-Ilan et al., 2005). Virulence is a measurable characteristic of the ability of the microorganism to cause disease and is intended for within-group or within-species comparisons.

Perhaps the best way to illustrate the difference between these two terms is to provide an example of each. The nematode–bacterium complex of Steinernema carpocapsae–Xenorhabdus nematophila shows pathogenicity to some non-insect arthropods (e.g., ticks) (Samish et al., 2000) but not to vertebrates (Akhurst and Smith, 2002). The fungus Beauveria bassiana GA strain is pathogenic to the pecan weevil, Curculio caryae, but B. bassiana MS1 strain is not as virulent to this host (Shapiro-Ilan et al., 2003). Finally, the Agrotis ipsilon nucleopolyhedrovirus (NPV) is more virulent to its original host, the black cutworm (Agrotis ipsilon) from which it was isolated, compared to Autographa californica NPV, which has a wide lepidopterous host range but is less virulent to Agrotis ipsilon (Boughton et al., 1999).

1.3.7 Dosage

One pathogen can infect and kill a host, as has been demonstrated with the infective juvenile of the nematode–bacterium complex (Steinernema–Xenorhabdus or Heterorhabditis–Photorhabdus) (Kaya and Gaugler, 1993). In general, however, with other entomopathogens, a minimal number of infective propagules is needed to pass through the portal of entry for infection to occur. This number is referred to as a dose that can be defined as the quantity of an active agent (i.e., entomopathogen) to which an insect is exposed at any one time. Dosage can be expressed quantitatively depending upon the host susceptibility in terms of mortality, infection, or time to death as lethal dose (LD), effective dose (ED), or lethal time to death (LT). The 50% or 90% level of response is assigned as LD50 or LD90, ED50 or ED90, or LT50 or LT90. The 50% level of response is referred to as the median lethal dose, median effective dose, or median lethal time. To obtain this type of information, a bioassay is conducted with a minimum of five dosage levels of the infective stage (i.e., occlusion bodies, spores, conidia, infective juveniles) plus a control treatment administered to a given stage of the host, and the response (i.e., mortality, infection, or time to death) is plotted with dosage on the x-axis and the response on the y-axis. If host mortality occurs in the control, Abbott’s formula can be used to correct for the mortality in the treatments (Abbott, 1925). Ideally, the range of the dosages should provide a response level between 10% and 90%. The lowest and highest dosages should not give a 0% or 100% response, respectively. The range of dosages obtained from the bioassay when plotted against the host response (i.e., mortality, infection, or time to death) should give an S-shaped curve which is transformed into a straight line by converting the response to a probit scale, and the dosage to the log scale. The level of response can be obtained by going up the probit scale on the y-axis and reading across to where the slope of the line intersects the dosage scale on the x-axis, which then provides the dosage for that response. The bioassay should be conducted at least twice with a different cohort of insects and entomopathogens to demonstrate that the data are reproducible. For further information on conducting bioassays with entomopathogens, the reader should refer to Burges and Thomson (1971) and Navon and Ascher (2000).

Dose is a precise number of the infective stage to which the bioassay insects are subjected to and usually attaining this level of precision is not possible. That is, it is not possible to determine the dosages quantitatively because of the size of the insect, the bioassay system (e.g., placing the infective stage on the diet surface and not knowing the amount of entomopathogen acquired), or because the insects live in an aqueous habitat where the dose cannot be calculated. In such cases, median concentration of the entomopathogen which produces a response in half of the test insects is used because the experimental method is not sufficiently accurate to determine the precise dose to which the test insects were exposed. For example, the median lethal concentration would then be expressed as LC50 or the median effective concentration would be EC50.

1.3.8 Signs, Symptoms, and Syndromes

Diseased insects exhibit characteristic aberrations or dysfunctions that are designated as signs and symptoms. When there is a physical or structural abnormality, the term sign is used, whereas when there is a functional or behavioral aberration, the term symptom is used. A sign is indicated by abnormalities in the morphology or structure such as color, malformed appendages or body segments, fragility of the integument, etc., whereas a symptom may be expressed by abnormal movement, abnormal response to stimuli, digestive disturbances (vomiting or diarrhea), inability to mate, etc.

A particular disease has a group of characteristics signs and symptoms which is called a syndrome. The syndrome refers to a system complex or a particular combination or sequence of signs and symptoms. Sometimes, the syndrome is very characteristic and specific for a disease caused by an entomopathogen. More often, the same syndrome occurs with many different diseases. Vomiting and diarrhea may develop from ingestion of chemical pesticides, bacterial toxins, or entomopathogenic bacteria, viruses or protistans. A distinct syndrome occurs when a silkworm (B. mori) larva ingests Bt subspecies sotto spores and δ-endotoxin. The larva stops feeding in a few minutes, becomes sluggish in about 10 min, the pH of the blood increases and the pH of the midgut decreases, and within an hour, the larva becomes moribund and dies.

1.3.9 Course of Infection

After an entomopathogen invades a healthy, susceptible insect, it starts to reproduce and the course of infection can be partitioned into various phases or stages: the incubation period, the beginning of disease with the appearance of the first signs and/or symptoms, and peak of disease (Fig. 1.3). The incubation period is the time from when the entomopathogen infects a host until the development of signs and/or symptoms. The appearance of the signs and/or symptoms indicates that there is a patent or frank infection including the production of toxins with some entomopathogens marking the beginning of disease. As the entomopathogen reproduces, the disease manifests itself to the fullest extent in the host reaching the peak of disease. The peak of the disease is when the signs and symptoms are most severe and either start to abate or attain a steady state. Also at this time, the entomopathogen has usually reached its highest level of reproduction, and if toxins are produced, they are present in the greatest amount. However, many entomopathogens, especially bacteria, fungi, and steinernematid and heterorhabditid nematodes, continue to develop and reproduce after the death of the host. When the peak of disease is reached, the insect may recover as the result of an immune response or die because of the absence of an immune response or the presence of only a weak immune response. In the case where the entomopathogen has low virulence, the insect may have a chronic infection and survive to adulthood and reproduce.

FIGURE 1.3 When a healthy, susceptible insect acquires an infectious concentration of an entomopathogen, it sets into motion a sequence of events referred to as the course and progress of disease. As the pathogen reproduces and/or produces toxins, the intensity of disease increases. The time from when the insect acquires the pathogen until its death is referred to as the period of lethal infection.

(Modified after Tanada and Kaya, 1993.)

The time from when the insect enters and infects the host until the insect dies is referred to as the period of lethal infection, or if the insect recovers as the period of infection. A short period of lethal infection indicates that the entomopathogen has high virulence, whereas a long period of lethal infection indicates that it has low virulence. An entomopathogen with high virulence is not necessarily the most infectious. For example, Bt subspecies kurstaki has high virulence to cabbage butterfly (Pieris rapae) larvae with a period of lethal infection of 48 h. But high virulence does not mean that the entomopathogen is highly contagious, and Bt subspecies kurstaki is not easily transmitted from one insect to another. Conversely, an entomopathogen with low virulence (i.e., period of lethal infection of several weeks as occurs with some microsporidian infections) may be highly contagious, with the pathogen being easily transmitted from one insect to another.

1.3.10 Acute, Chronic, and Latent Infections

Infections in insects may vary from latent to chronic to acute. Acute infections are the most apparent because of the distinct and characteristic response of the insect to the pathogen causing the disease. Acute infections are of short duration and usually result in the death of the host (i.e., the period of lethal infection is short). Chronic infections are often overlooked because their manifestations tend to be less striking and apparent to the observer until very late in the infection process. Chronic infections are of long duration and the hosts may or may not die. If the host dies, the period of lethal infection is long, whereas if the host does not die, it may reach adulthood and reproduce. Latent infections in insects have been detected primarily with viruses. In such cases, the term latent or occult viral infection is used, and the virus is referred to as an occult virus and not as a latent virus (Onstad et al., 2006). Recently, an occult nodavirus belonging to the genus Alphanodavirus in the family Nodaviridae was described from an insect cell line (Li et al., 2007).

1.3.11 Koch’s Postulates

One of the basic tenets in pathology for establishing the etiological or causal agent of a disease involving microorganisms is the application of Koch’s postulates. Robert Koch (1843–1910), a German physician who is considered one of the founders of microbiology, made brilliant discoveries on the causal agents of anthrax, tuberculosis, and cholera through the application of postulates that bear his name.

Koch’s postulates as stated in Tanada and Kaya (1993) are:

1 A specific pathogenic organism must be seen in all cases of a disease.

2 This organism must be attained in pure culture.

3 The organism from the pure culture must reproduce the disease in experimental animals.

4 The same organism must be recovered from the experimental animal.

It is not always possible to fulfill Koch’s postulates because one or more steps cannot be performed or can be performed only with great difficulty. For step 1, some organisms are difficult to detect, although the electron microscope has made it possible to view submicroscopic organisms such as viruses, rickettsia, rickettsiella, and mollicutes putatively in infected insects. However, the mere presence of these organisms in a host does not prove that they are the causal agent of a particular disease without further experimentation by following Koch’s postulates. For step 2, certain microorganisms cannot be isolated in pure culture with the present available laboratory methods or are very difficult to culture. These microorganisms are mainly obligate pathogens requiring living cells or tissues. For some obligate microorganisms, insect tissue cultures can be used to fulfill step 2. For step 3, different pathogens may produce similar signs, symptoms, and syndromes which may mistakenly result in designating the wrong organism as the etiological agent.

The contributions of molecular biology have opened up the concept of molecular Koch’s postulates to help characterize whether a particular microbial gene is an essential constituent of the ability of a microorganism to infect and cause disease in a given host (Falkow, 1988). Recently, Falkow (2004) summarized the application of the molecular Koch’s postulates to bacterial pathogenicity in animal systems. Table 1.1 shows Koch’s original three postulates with the proposed molecular Koch’s postulates (Falkow, 2004). Koch’s original three postulates are essentially the same four steps as stated by Tanada and Kaya (1993). The molecular Koch’s postulates are used to demonstrate that a particular gene or set of genes is the virulence factor and inactivation of this gene(s) results in the loss of pathogenicity. Restoration of pathogenicity should occur with the reintroduction of the gene into the microorganism. To the authors’ knowledge, the molecular Koch’s postulates as proposed by Falkow (2004) are not intentionally used in insect pathology. However, the insecticidal protein genes of Bt have been cloned into other Bt subspecies or other bacterial species and have proven to be efficacious against the target insects (Park and Federici, 2009).

TABLE 1.1. Comparison of Koch’s Original Postulates and the Molecular Koch’s Postulates